Methods and devices for preparation of lipid nanoparticles

ABSTRACT

The present invention provides for a process for preparing liposomes, lipid discs, and other lipid nanoparticles using a multi-port manifold, wherein the lipid solution stream, containing an organic solvent, is mixed with two or more streams of aqueous solution (e.g., buffer). In some aspects, at least some of the streams of the lipid and aqueous solutions are not directly opposite of each other. Thus, the process does not require dilution of the organic solvent as an additional step. In some embodiments, one of the solutions may also contain an active pharmaceutical ingredient (API). This invention provides a robust process of liposome manufacturing with different lipid formulations and different payloads. Particle size, morphology, and the manufacturing scale can be controlled by altering the port size and number of the manifold ports, and by selecting the flow rate or flow velocity of the lipid and aqueous solutions.

This application claims priority to U.S. provisional patent applicationNo. 61/791,054, filed on Mar. 15, 2013, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present technology generally relates to liposomes and, morespecifically, to liposomes encapsulating an active pharmaceuticalingredient.

BACKGROUND

Liposome technology has been utilized for drug delivery in clinicaltherapy and scientific research. The current methods for liposomepreparation are used largely for small-scale laboratory research.Exemplary methods include a lipid dry film rehydration/extrusion method,a detergent dialysis method, and an ethanol evaporation and dilutionmethod.

U.S. Pat. No. 7,901,708 and US Patent Publication No. 2007/0042021,incorporated herein by reference, refer to a two-step method forliposome preparation: (i) using a T-connector to mix a lipid-organicsolvent solution with an aqueous solution; (ii) diluting the mixturewith an aqueous solution.

The currently available methods present difficult problems associatedwith scalability, low reproducibility and product heterogeneity. Thereexists a need for improved methods to make liposomes for use in drugdelivery.

SUMMARY OF THE INVENTION

The present invention provides a method for preparing lipidnanoparticles (LNP). In preferred aspects, the method comprises:

a) introducing i) one or more streams of a lipid solution via a firstset of one or more inlet ports of a manifold and ii) one or more streamsof an aqueous solution via a second set of one or more an inlet ports ofthe manifold, thereby mixing the lipid solution and the aqueous solutionso as to produce an LNP solution; and

b) recovering the LNP solution via one or more outlet ports of themanifold. In the above method, the angle between at least one lipid andat one aqueous solution inlet ports is not 180° or a substantiallysimilar angle. In other words, at least one stream of lipid solution andat one stream of aqueous solution collide at an angle less than about180°. Thus, in some aspects, the method does not include a T-connector.

The invention also provides the LNP solution made by the above method, apharmaceutical composition prepared using the LNP solution.

The invention further provides a device adapted to perform the method,such as a manifold system described in detail below.

The present invention also provides a method for producing LNPcontaining an active pharmaceutical ingredient (“API”), wherein suchAPI-containing LNP are produced in a single mixing step.

According to exemplary embodiments, the present invention provides adevice for preparing liposomes encapsulating an API that includes amanifold that may have a mixing chamber, at least one lipid solutioninlet port connected to the chamber; and a plurality of aqueous solutioninlet ports connected to the chamber.

Another embodiment of the invention provides for a process for preparingliposomes that encapsulate an active pharmaceutical ingredient (API)that may include a step of providing, (i) a lipid solution that mayinclude an organic solvent and a lipid, in a lipid solution reservoir,and (ii) an aqueous solution comprising water and a buffer, in anaqueous solution reservoir; and a step of providing a manifold that thatmay include (i) a mixing chamber; (ii) at least one lipid solution inletport connected to the chamber; and, (iii) a plurality of aqueoussolution inlet ports connected to the chamber; a step of mixing thelipid solution and the aqueous solution, as a stream of each solution isintroduced into the mixing chamber, to produce liposomes; and a step ofencapsulating the active pharmaceutical ingredient within the liposomes.

In an alternative embodiment, the invention provides liposomes made bythe process of the invention.

The invention also provides liposomes, wherein the liposomes encapsulatean API, and the average diameter of the liposome is about 10-300 nm.

This invention enables control of LNP size, size distribution,morphology, and manufacturing scale by altering the port size, numberand geometry of the manifold, and by selecting the flow rate or flowvelocity of the lipid and aqueous solutions. These and other advantagesof the present technology will be apparent when reference is made to theaccompanying drawings and the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B illustrate projections from the top (FIG. 1A) andside (FIG. 1B) of an exemplary 5-port manifold for preparing liposomesencapsulating an API.

FIG. 2 shows a flowchart of an exemplary process for preparing liposomesencapsulating an API.

FIG. 3 is schematic representation of an embodiment of the invention inwhich an insoluble API is encapsulated with liposomes made with a 5-portexemplary manifold.

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D show the effect of flow rate andmanifold pore size on liposome particle size for a 5-port manifold withpore sizes of 1 mm (FIG. 4A and FIG. 4B) and 1.6 mm (FIG. 4C and FIG.4D).

FIG. 5 is a Cryo-TEM image of liposomes made with a 5-port exemplarymanifold and loaded with doxorubicin.

FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D show the effect of flow rate andmanifold pore size on the size (FIG. 6A and FIG. 6C), polydispersityindex (FIG. 6B and FIG. 6D) of siRNA liposomes, and Cryo-TEM image (FIG.6E) of the liposomes.

FIG. 7A shows a Cryo-TEM image of unilamellar liposomes generated at aflow rate of 40 ml/min.

FIG. 7B shows a Cryo-TEM image of lipid discs generated at a flow rateof 5 ml/min.

FIG. 8A shows alternative exemplary embodiments for the number andorientation of inlets and outlets in a 7-port manifold of the invention,with 3 outlet ports. FIG. 8B shows alternative exemplary embodiments forthe number and orientation of inlets and outlets in a 7-port manifold ofthe invention, with 2 outlet ports. FIG. 8C and FIG. 8D show alternativeexemplary embodiments for the number and orientation of inlets andoutlets in a 7-port manifold of the invention, with 1 outlet port.

DETAILED DESCRIPTION

While this invention is susceptible of embodiments in many differentforms, there are shown in the drawings and will herein be described indetail several specific embodiments with the understanding that thepresent disclosure is to be considered as an exemplification of theprinciples of the technology and is not intended to limit the inventionto the embodiments illustrated.

DEFINITIONS

The term “flow rate” refers to the volume of a lipid solution or anaqueous solution fed to an inlet port.

Term “flow velocity” refers to the liquid flow speed in the inlet port,for example, calculated as V=R/6000 A, where V (m/s) is the flowvelocity, R (ml/min) is the flow rate, A (cm²) is the cross section areaof the pore of an inlet port.

The term “lipid nanoparticles,” or LNP, refers to liposomes (e.g.,unilamellar or multilamellar), solid lipid particles or lipid discs.Exemplary liposomes and lipid discs are shown in the Examples.

The term of “cationic lipid” refers to a lipid that carries a netpositive charge at about pH 3-pH 9.

As used herein the term of “anionic lipid” refers to a lipid or acholesterol derivative that carries a net negative charge at about pH3-pH 9.

The term “pegylated lipid” refers to a lipid that is conjugated with apolyethylene glycol polymer.

The term “neutral lipid” refers to the lipid that does not carry netcharge at about pH 3-pH 9.

The term of “lipid-anchored molecule” refers to a molecule that has alipid or cholesterol anchor and thus may be incorporated into aliposome.

The term of “active pharmaceutical ingredient” or API refers to apharmaceutical active ingredient that is used for disease treatment orfor disease prevention (vaccine). API may also refer to an ingredientintended for disease diagnosis.

FIG. 1 A and FIG. 1B, illustrate an embodiment of the invention. Afive-port manifold is shown having one lipid solution inlet port, threeaqueous solution inlet ports and one liposome outlet port. FIG. 1 A, isa projection from the top of the manifold, while FIG. 1 B, is aprojection from the side of the manifold. FIG. 1A and FIG. 1B show thatmanifold mixing chamber 110 is connected to one lipid solution inletport 120, through which the lipid solution enters the mixing chamber.Three aqueous solution inlet ports 130 are also connected to the mixingchamber, and provide passage of the aqueous solution to enter thechamber. This figure illustrates that lipid solution inlet port and theaqueous solution inlet ports may have an inner diameter indicated by140. Lipid solution 150 enters into the lipid solution inlet port whileaqueous solution 160 passes into the aqueous solution inlet ports andthe LNP 180 exit LNP outlet port 170.

FIG. 2 is a flow chart that illustrates an exemplary process that may beused to implement an embodiment of the present technology. As shown inFIG. 2, the flow chart provides for lipid solution 210 that includes anorganic solvent and a lipid, and aqueous solution 220 that includeswater and a buffer. The lipid solution or the aqueous solution mayfurther include a solubilized API. The lipid solution and the aqueoussolution may simultaneously enter the mixing chamber of manifold 230. Insome embodiments one, or the other or both solutions have a positivepressure, which may be provided by a pump apparatus. The lipid solutionand the aqueous solution are mixed in the mixing chamber to produce asolution of LNP 240. The process of the invention also provides for astep of encapsulating the API within the LNP. When an API is solubilizedin either the lipid solution or the aqueous solution, the step ofencapsulating the API may occur during formation of the LNP in themixing chamber. In other embodiments, the API may be incorporated intothe LNP by diffusion of the agent from outside the liposome. In someembodiments, the methods of the invention comprises step c) of loadingLNP recovered from the LNP solution with an API.

FIG. 3 shows a schematic representation of one embodiment of theinvention in which a water insoluble API is dissolved in the lipidsolution. An aqueous solution in reservoir 310 may be conveyed tomanifold 370 through conduits 330. Simultaneously, a lipid solutioncontaining the solubilized API in reservoir 340 maybe conveyed tomanifold 370, through conduit 360. Pumps 320 and 350 may be used toadjust and monitor the flow rate of each solution. The aqueous solutionenters the manifold through the aqueous solution inlet ports, and thelipid solution enters the manifold through the lipid solution inletport, shown previously in FIG. 1A and FIG. 1B. The mixing of the aqueousand the lipid solution in the manifold results in the formation of lipidnanoparticles 380 which exit the manifold through the lipid nanoparticleoutlet port, as shown in FIG. 1A and FIG. 1B.

FIG. 4 A and FIG. 4B show results for liposomes containing doxorubicinHCl manufactured using a 5-port manifold having inlet and outlet ports 1mm in diameter. At a flow rate of 1 ml/min the liposomes have an averagediameter of about 150 nm. As the flow rate increases, the diameter ofthe liposomes decreases, until it reaches a plateau of about 50 nm whenthe flow rate is from 20 to 50 ml/min. Graph B shows that thepolydispersity index (“PDI”), a measure of particle size distribution,is about 0.2 at a flow rate from 20 to 50 ml/min. The PDI increases toabout 0.3 when the flow rate is below 10 ml/min. FIG. 4 C and FIG. 4Dshow results for liposomes manufactured with a 5-port manifold havingports of 1.6 mm in diameter. Panel C shows that at a flow rate of 5ml/min, the liposomes have an average diameter of about 120 nm. There isan approximately linear decrease in diameter as the flow rate increasesup to 60 ml/min. Panel D shows that the PDI of 0.35 is relativelyindependent of flow rate.

FIG. 5 shows Cryo-TEM imaging of liposomes loaded with doxorubicin. Theliposome was made by a 5-port manifold having ports 1.0 mm in diameter.The formulation is substantially the same as Doxil which is a clinicallyused formulation of the anticancer liposome drug, doxorubicin. As shownin the figure, lipids form unilamellar liposomes, in which doxorubicinforms crystals inside.

FIG. 6 A, FIG. 6B, FIG. 6C and FIG. 6D show the results obtained forliposomes containing siRNA. FIG. 6 A and FIG. 6B illustrate the resultsfor liposomes manufactured with a 5-port manifold having ports 1 mm indiameter. At a flow rate of 5 ml/min the liposomes have an averagediameter of about 160 nm. The diameter of the liposomes decreases whenthe flow rate is 20 ml/min and does not change substantially as the rateis further increased. The PDI of 0.008 is relatively independent of flowrate. FIG. 6C and FIG. 6D illustrate the results for liposomesmanufactured with a 5-port manifold having ports 1.6 mm in diameter.FIG. 6C shows that a flow rate of 5 ml/min the liposomes have an averagediameter of about 150 nm. The size decreases to about 90 nm when theflow rate increases to 10 ml/min. A further increase in flow rate doesnot result in a substantial change in the size of the nanoparticle. FIG.6D shows the PDI is about 0.1-0.2 in the range of flow rate from 5 to 50ml/min. FIG. 6E shows the Cryo-TEM images of siRNA liposomes. As can beseen from the figure, the particle size is homogenous, while themorphology is not unilamellar or multilamellar.

FIGS. 7A-7B show the effect of flow rate on the morphology of the lipidparticles. FIG. 7A shows a Cryo-TEM image of unilamellar liposomesgenerated at a flow rate of 40 ml/min (pore size 1 mm; 5-port manifold),which produced particle with a diameter of 81.1 nm and PDI of 0.021. Thecrystals inside the liposomes are the loaded doxorubicin. FIG. 7B showsa Cryo-TEM image of lipid discs generated at a flow rate of 5 ml/min(pore size 1 mm; 5-port manifold), which produced predominantlygenerated lipids discs with about 60 nm in diameter and about 6 nm inlipid bilayer thickness.

FIGS. 8A-8D show alternative exemplary embodiments for the number andorientation of inlets and outlets in a 7-port manifold of the invention,which was used in Example 8. Arrows indicate the direction of flow inthe ports, while the blunted lines indicate sealed unused ports in aprefabricated manifold.

A Device for Preparing Liposomes Encapsulating an Active PharmaceuticalAgent

The invention provides a device adapted to perform the method of theinvention, such as a manifold system described herein.

In some aspects, the present technology provides a device for preparingLNP encapsulating an API that includes a manifold that may have a mixingchamber, at least one lipid solution inlet port connected to thechamber; and a plurality of aqueous solution inlet ports connected tothe chamber.

In a preferred embodiment, the device may include a LNP solution outletport connected to the chamber.

Preferably, the device may include a reservoir for a lipid solutionwhich is connected to the lipid solution inlet port by a lipid solutionconduit, and a reservoir for an aqueous solution which is connected tothe aqueous solution inlet ports by an aqueous solution conduit.

The inlet ports for the lipid and aqueous solutions, and the exit portfor the liposome solutions may have an internal diameter which is thesame or different. Preferably inlet and outlet ports have an internaldiameter from about 0.1 mm to about 10 mm. More preferably the portshave an internal diameter from about 0.15 mm to about 5 mm.

In some embodiments, the mixing chamber is located at the point ofconversion of the conduits and may itself be formed by two or moreconduits passing through each other, or intersecting, without any changein the shape of the conduits. For instance, the mixing chamber canformed by drilling in a solid material two or more pass-through channelsall intersecting at the point of conversion. In addition, one or moreconduits may be sealed so that there is no passage of fluid permittedthrough the conduit. Such seal may be located either immediately priorto the point of intersection or distantly therefrom. For example, sealedconduits are illustrated in FIGS. 8A, 8C and 8D.

In some embodiments, one manifold may contain more than one mixingchamber. For example, one set of inlet ports intersect at one chamber,and another group of inlet ports intersect at another chamber, and thetwo chambers are connected by conduits to the third mixing chamber thatis connected to an outlet port.

Preferably, a pump is used to induce a positive flow to the lipidsolution and to the aqueous solution. The pump may be an inline pump ora syringe pump.

Typically, the mixing chamber may be connected to 2 to about 20 aqueoussolution inlet ports. Preferably, there may be from 3 to about 11 suchports, from 3 to about 12 such ports. More preferably, there are from 3to about 10, or from 3 to about 7 aqueous solution entry inlet ports.The mixing chamber may also be connected to from 1 to about 5 lipidsolution inlet ports. Preferably, there are from 1 to about 3 lipidsolution inlet ports. Most preferably, there is 1 or 2 lipid solutioninlet ports. In a preferred embodiment, the mixing chamber is connectedto at least 1 (e.g., 1, 2, 3, 4, or 5) lipid solution port(s) and atleast 2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20) aqueous solution inlet ports.

The mixing chamber is further connected to 1 to about 3 outlet ports forthe liposome solution for particle size control, preferably, there is 1(e.g., 1, 2, or 3) outlet port(s).

In certain aspects, the angle between the inlet ports for the lipid andaqueous solutions is from about 18° to about 180°. Preferably the angelmay be from about 24° to about 180°, more preferably from about 30° toabout 180°. In some embodiments, the angle between at least one lipidand at one aqueous solution inlet ports is not 180° or a substantiallysimilar angle. For example, the angle between at least one lipid and atone aqueous solution inlet ports is about 120° or less, about 90° orless, for example, as shown in FIGS. 8A-8D. The angle between ports isthe angle at which streams of respective solutions are directed into themixing chamber.

The lipid and aqueous solutions may have the same flow rate through themanifold. Alternatively, the solutions may have different flow rates.The flow rates for the lipid and aqueous solutions may be 1 ml/min toabout 6,000 ml/min, e.g., from about 1 ml/min to about 1,500 ml/min.Preferably, the flow rates may be from about 5 ml/min to about 1,000ml/min, e.g., from about 5 ml/min to about 400 ml/min. More preferably,the rates may be the rates may be from about 20 ml/min to about 600ml/min or from about 10 ml/min to about 300 ml/min. In some embodiments,the flow rates are adjusted based on the size of inlet ports to obtainthe desired LNP size, morphology, PDI, and manufacturing scale.

Process for Preparing LNP

The invention provides a method for preparing lipid nanoparticles (LNP),the method comprising:

a) introducing i) one or more streams of a lipid solution via a firstset of one or more inlet ports of a manifold and ii) one or more streamsof an aqueous solution via a second set of one or more an inlet ports ofthe manifold, thereby mixing the lipid solution and the aqueous solutionso as to produce an LNP solution; and

b) recovering the LNP solution via one or more outlet ports of themanifold;

wherein the angle between at least one lipid and at one aqueous solutioninlet ports is not 180° or a substantially similar angle. In someaspects, at least one stream of lipid solution and at one stream ofaqueous solution collide at an angle less than about 180°. Thus, in someaspects, the method does not include a T-connector.

In some embodiments, the angle between at least one lipid and at oneaqueous solution inlet ports is about 120° or less, e.g., 115° or less,100° or less, 90° or less, 80° or less, 72° or less, 60° or less, 45° orless, 30° or less, 18° or less,

In some embodiments, the aqueous solution in step ii) is introduced viaat least two inlet ports, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In some embodiments, theaqueous solution in step ii) is introduced via at least 3 but no morethan 11 inlet ports, e.g., at least 3 but not more than 7, at least 3but no more than 5, at least 4 but no more than 11, at least 5 but nomore than 11, at least 6 but no more than 11.

In some embodiments, at least two (e.g., 3, 4, 5, 6, 7, etc.) aqueousinlet ports and at least one (e.g., 2, 3, 4, 5, etc.) lipid solutioninlet port are in the same plane.

In some embodiments, at least one (e.g., 2) outlet port is substantiallyperpendicular to the plane of inlet ports. In other embodiments, atleast one (e.g., 2, 3, 4, 5, etc.) outlet port is substantially notperpendicular to the plane of inlet ports.

In some embodiments, at least two (e.g., 3, 4, 5, 6, 7, etc.) aqueoussolution inlet ports and at least one (e.g., 2, 3, 4, 5, etc.) lipidsolution inlet port are not in the same plane.

In some embodiments, the aqueous solution introduced into at least oneof the inlet ports differs from a second aqueous solution introducedinto another inlet port.

In some embodiments, the aqueous solution and/or the lipid solutioncomprises an active pharmaceutical ingredient (API).

In some embodiments, step a) further comprises introducing iii) one ormore streams of non-aqueous solutions via one or more inlet ports of themanifold.

Another embodiment of the invention provides for a process for preparingLNP that encapsulate an active pharmaceutical ingredient (API) that mayinclude a step of providing (i) a lipid solution that may include anorganic solvent and a lipid, in a lipid solution reservoir, and (ii) anaqueous solution comprising water and a buffer, in an aqueous solutionreservoir; and a step of providing a manifold that that may include (i)a mixing chamber; (ii) at least one lipid solution inlet port connectedto the chamber; and, (iii) a plurality of aqueous solution inlet portsconnected to the chamber; a step of mixing the lipid solution and theaqueous solution, as a stream of each solution is introduced into themixing chamber, to produce LNP; and a step of encapsulating the activepharmaceutical ingredient within the LNP.

In one embodiment of the process, the lipid solution may include the APIto be encapsulated. In another embodiment, the aqueous solution mayinclude the API.

The step of encapsulating the drug into a liposome may occur at the sametime as the mixing step when the drug is solubilized in the lipid oraqueous solution. While not being bound by theory it is believed thatthe LNP form instantly when the aqueous solution and the lipid solutionmake contact. An API, carried by the lipid solution or by the aqueoussolution, may be encapsulated in the LNP through either lipophilicinteraction, or electrostatic interaction, or both, between the API andthe lipids.

Alternatively the API may be introduced into empty LNP by a diffusion oranother loading process as illustrated in FIG. 2.

An exemplary manifold of the process is described above and shown inFIG. 1A and FIG. 1B

The Lipid and Aqueous Solutions

The invention utilizes lipid and aqueous solutions. The lipid solutionmay comprise an organic solvent. The organic solvent may be a watermiscible solvent. Preferably, the water miscible solvent is selectedfrom the group consisting of ethanol, methanol, DMSO and isopropanol.Most preferably, the organic solvent is ethanol.

The lipid solution may include a mixture of lipids. The mixture oflipids preferably includes cholesterol.

The mixture of lipids may also include a cationic lipid. The cationiclipid may be, but is not limited to, N,N-dioleyl-N,N-dimethylammoniumchloride (“DODAC”); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride (“DOTMA”); N-(2,3-dioleyloxy)propyl)-N,N-dimethylammoniumchloride (“DODMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);N-(2,3-dioleoyloxy)propyl)-N,N-dimethylammonium chloride (“DODAP”);3-(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (“DC-Choi”);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”); 1.2-dilinoleyloxy-N,N-dimethyl-3-aminopropane(DLinDMA); 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA);1.2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA);2-{4-[(3b)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-amine(CLinDMA).

In some embodiments the mixture of lipids may include an anionic lipid.The anionic lipid may be but are not limited to diacylglycerolphophatidic acid (1,2-distearoyl-sn-glycero-3-phosphate (DSPA);1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA);1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA);1,2-dilauroyl-sn-glycero-3-phosphate (DLPA);1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)), diacylglycerolphosphoglycerol (1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol)(DSPG); 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG);1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG);1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DLPG);1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG)),phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines,lysylphosphatidylglycerols, and other anionic modifying groups joined toneutral lipids. The mixture of lipids may also include a neutral lipid.The neutral lipids may be but are not limited to diacylglycerolphosphocholine (L-α-phosphatidylcholine, hydrogenated (Soy) (HSPC);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC);1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylglycerolphosphoethanolamine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine(DSPE); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE);1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE);1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE);1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), andphosphatidylserine.

The mixture of lipids may also include a pegylated lipid. The pegylatedlipid may be but are not limited to1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DSPE);1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DOPE);1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DPPE);1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DMPE);1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DLPE);1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DSPE);1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DOPE);1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DPPE);1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DMPE);1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DLPE).

The mixture of lipid may also include a lipid-like molecule or lipidoid.The mixture of lipid may also include a lipid- or cholesterol-conjugatedmolecule including a protein, or a peptide, or an oligonucleotide.

The aqueous solution of the process preferably includes water and abuffer. Buffers may be of but are not limited to phosphate, histidine,HEPES, Tris, acetate, and citrate.

Active Pharmaceutical Ingredient

Preferably, the API may be an anticancer agent, an anti-inflammatoryagent, and an anti-diabetic agent, an anti-fungal agent and anantibiotic agent.

The API may be a polynucleotide (including an oligonucleotide) a proteinor a small molecule.

In one embodiment the API is a polynucleotide. The polynucleotide may bea genomic DNA fragment, cDNA, mRNA, ssRNA, dsRNA, microRNA, siRNA,shRNA, sdRNA, DsiRNA, LNA, and antisense DNA or RNA.

Alternatively, the API may be a small organic molecule API. Preferably,the molecule has a molecular weight from about 1500 g/mole to about 50g/mole.

An API can be, for example, an anticancer agent, an antibiotic agent, anantiviral agent, an anti-fungal agent, or an analgesic.

Exemplary anticancer agents that may include but are not limitedacivicin, aclarubicin, acodazole, ametantrone, aminoglutethimide,anthramycin, asparaginase, azacitidine, azetepa, bisantrene, bleomycin,busulfan, cactinomycin, calusterone, caracemide, carboplatin,carmustine, carubicin, chlorambucil, cisplatin, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, daunorubicin, dezaguanine,diaziquone, docetaxel, doxorubicin, epipropidine, erlotinib, etoposide,etoprine, floxuridine, fludarabine, fluorouracil, fluorocitabine,hydroxyurea, iproplatin, leuprolide acetate, lomustine, mechlorethamine,megestrol acetate, melengestrol acetate, mercaptopurine, methotrexate,metoprine, mitocromin, mitogillin, mitomycin, mitosper, mitoxantrone,mycophenolic acid, nocodazole, nogalamycin, oxisuran, paclitaxel,peliomycin, pentamustine, porfiromycin, prednimustine, procarbazinehydrochloride, puromycin, pyrazofurin, riboprine, semustine,sparsomycin, spirogermanium, spiromustine, spiroplatin, streptozocin,talisomycin, tegafur, teniposide, teroxirone, thiamiprine, thioguanine,tiazofurin, triciribine phosphate, triethylenemelamine, trimetrexate,uracil mustard, uredepa, vinblastine, vincristine, vindesine,vinepidine, vinrosidine, vinzolidine, zinostatin and zorubicin.

Exemplary antibiotic agents that may include but are not limited toaminoglycoside; amikacin; gentamicin; kanamycin; neomycin; netilmicin;steptomycin; tobramycin; ansamycins; geldanamycin; herbimycin;carbacephem; loracarbef; carbacepenem; ertapenem; doripenem;imipenem/cilastatin; meropenem; cephalosporin; cefadroxil; cefazolin;cefalotin or cefalothin; cefalexin; cefaclor; cefamandole; cefoxitin;cefprozil; cefuroxime; cefixime; cefdinir; cefditoren; cefoperazone;cefotaxime; cefpodoxime; ceftazidime; ceftibuten; ceftizoxime;ceftriaxone; cefepime; ceftobiprole; glycopeptide; teicoplanin;vancomycin; macrolides; azithromycin; clarithromycin; dirithromycin;erythromicin; roxithromycin; troleandomycin; telithromycin;spectinomycin; monobactam; aztreonam; penicillins; amoxicillin;ampicillin; azlocillin; carbenicillin; cloxacillin; dicloxacillin;flucloxacillin; mezlocillin; meticillin; nafcillin; oxacillin;penicillin, piperacillin, ticarcillin; bacitracin; colistin; polymyxinB; quinolone; ciprofloxacin; enoxacin; gatifloxacin; levofloxacin;lomefloxacin; moxifloxacin; norfloxacin; ofloxacin; trovafloxacin;sulfonamide; mafenide; prontosil (archaic); sulfacetamide;sulfamethizole; sufanilimide (archaic); sulfasalazine; sulfisoxazole;trimethoprim; trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX);tetracycline; demeclocycline; doxycycline; minocycline; oxytetracycline;tetracycline; arsphenamine; chloramphenicol; clindamycin; lincomycin;ethambutol; fosfomycin; fusidic acid; furazolidone; isoniazid;linezolid; metronidazole; mupirocin; nitrofuantoin; platensimycin;purazinamide; quinupristin/dalfopristin; rifampin or rifampicin; andtimidazole. In specific embodiments, the anti-cancer agent is chosenfrom daunorubicin, doxorubicin, paclitaxel, docetaxel, cisplatin,carboplatin, cytarabine, floxuridine, fludarabine, fluorouracil,iproplatin, leuprolide acetate, and methotrexate.

Exemplary antiviral agent that may include, but are not limited tothiosemicarbazone; metisazone; nucleoside and/or nucleotide; acyclovir;idoxuridine; vidarabine; ribavirin; ganciclovir; famciclovir;valaciclovir; cidofovir; penciclovir; valganciclovir; brivudine;ribavirin, cyclic amines; rimantadine; tromantadine; phosphonic acidderivative; foscamet; fosfonet; protease inhibitor; saquinavir;indinavir; ritonavir; nelfinavir; amprenavir; lopinavir; fosamprenavir;atazanavir; tipranavir; nucleoside and nucleotide reverse transcriptaseinhibitor; zidovudine; didanosine; zalcitabine; stavudine; lamivudine;abacavir; tenofovir disoproxil; adefovir dipivoxil; emtricitabine;entecavir; non-nucleoside reverse transcriptase inhibitor; nevirapine;delavirdine; efavirenz; neuraminidase inhibitor; zanamivir; oseltamivir;moroxydine; inosine pranobex; pleconaril; and enfuvirtide.

Exemplary anti-fungal agent that may include but are not limited toallylamine; terbinafine; antimetabolite; flucytosine; azole;fluconazole; itraconazole; ketoconazole; ravuconazole; posaconazole;voriconazole; glucan synthesis inhibitor; caspofungin; micafungin;anidulafungin; polyenes; amphotericin B; amphotericin B ColloidalDispersion (ABCD); and griseofulvin.

Exemplary analgesics may include, but are not limited to opiatederivative, codeine, meperidine, methadone, and morphine.

LNP

The invention also embraces LNP made by the process described belowwherein the LNP encapsulate an API.

Preferably, more than 70% of API is encapsulated in the LNP. Morepreferably, more than 80% of API is encapsulated in the LNP, mostpreferably, more than 90% of API is encapsulated in the LNP.

Optionally, liposomes may be of unilamellar. Alternatively, theliposomes may be of multilamellar, or of inverted hexagonal or cubicmorphology, or as lipid discs, hollow liposomes, or solid lipidparticles.

The mean particle size of LNP made by the process is from about 10 nm toabout 2,000 nm, preferably less than 300 nm, more preferably, the meanparticle size may be about 10 to 300 nm or about 20 to about 300 nm.Most preferably, the mean particle size is about 20 to 120 nm or about30 to about 200 nm, most preferably, between about 30 and about 120 nm,about 10 and 120 nm, about 60 and about 100 nm, or 20 to about 80 nm.

In some embodiments, the LNP solution comprises substantially lipiddiscs. In other embodiments, the LNP solution comprises substantiallyliposomes.

In some embodiments, the LNP have a polydispersity index from about0.005 to about 0.8, e.g., 0.005 to about 0.5, 0.01 to about 0.5, 0.01 toabout 0.4, 0.01 to 0.2.

Methods for Making Liposome Solutions

Lipid Solution

The lipid solution may be made from the stock solutions of individuallipids that are mixed together. Lipids are preferably dissolved in anorganic solvent to make a lipid solution. The organic solvent used formaking the lipid solution may be miscible with water. Preferably thesolvent may ethanol, methanol, DMSO, propanol, DMF, THF, acetone,dioxane, ethylene glycol, polyethylene glycol and isopropanol. Morepreferably, the solvent is polyethylene glycol, isopropanol, andethanol. Preferably, the solvent includes less than 10% water. In somecases, the lipid solution may be made from a mixture of lipids,thereupon dissolving the mixture in an organic solvent. Theconcentration of the total lipids in the solution may be in the rangefrom about 1 mg/ml to about 200 mg/ml, e.g., from about 1 mg/ml to about100 mg/ml. More preferably, the concentration of the total lipids in thesolution may be in the range from about 5 mg/ml to about 80 mg/ml orform about 10 mg/ml to 100 mg/ml. In some embodiments, the organicsolvent is ethanol at a concentration of about 70% or more (e.g., 75% ormore, 80% or more, 85% or more, 90% or more, 95% or more, 100%).

The mixture of lipids will be optimized as required for optimal deliveryof the API and is readily optimized by routine experimentation by one ofordinary skill in the art. In Example 2 below, the total lipidconcentration of the Lipid Solution is 29 mg/ml; the lipids aredissolved in anhydrous ethanol.

In certain embodiments, a water-insoluble API may be dissolved in thelipid solution. The concentration of the API in the lipid solution willdepend on the efficacy of the agent and may easily be determined by oneof ordinary skill in the art. The lipid/API ratio will determined by theencapsulation power of the LNP to the API.

Preparing an Aqueous Solution with an API (S1)

A water-soluble API may be dissolved in a first aqueous solution (S1).The pH and salinity of the solution may be optimized to accommodate therequirements for the interaction between the API and the lipids to formliposome. These conditions may be readily determined by one of ordinaryskill in the art. As shown below in the examples, S1 in Example 6comprises 20 mM citrate, 0.5 mg/ml of siRNA, pH 5.0. The acidic pHprotonates lipid DLinDMA, and the positively charged lipid interact withthe negatively charged siRNA to encapsulate siRNA into liposomes. InExample 1, solutions 1, 2, and 3 are the solution of 250 mM (NH₄)₂SO₄,pH 6.5.

Preparing an Aqueous Solution without a API (S2)

As will be readily apparent to those of skill in the art, an aqueoussolution that lacks an API, referred to as (S2), may be similar to asolution having the agent. Alternatively, S1 and S2 may be different. Asshown in Example 6, S2 is a solution of 20 mM citrate and 100 mM NaCl,pH 5.0, while S1 is a solution of 20 mM citrate, pH 5.0.

Liposome Preparation

Mixing the Solutions

The lipid solution and the aqueous solution(s) preferably enter themanifold from different ports, each with a flow rate of from about 1ml/min to about 6000 ml/min. Preferably, the flow rates may be fromabout 5 ml/min to about 1000 ml/min. More preferably, the rates may befrom about 20 ml/min to about 600 ml/min. In some embodiments, the flowrates are adjusted based on the size of inlet ports to obtain thedesired LNP size, morphology, PDI, and manufacturing scales.

In some embodiments, the lipid solution and/or the aqueous solution isintroduced via port size of 0.1-0.5 mm at a flow rate about 1 ml/min toabout 2,500 ml/min.

In some embodiments, the flow velocity of the lipid solution and/or theaqueous solution is from about 0.002 m/s to about 10 m/s, e.g., from0.02 m/s to 8 m/s, from 0.2 m/s to 6 m/s. The flow velocity is adjustedbased on the size of inlet ports to obtain the desired LNP size,morphology, PDI, and manufacturing scale.

Loading of the API into LNP

By Solution Mixing

In the mixing chamber the lipids are believed to instantaneouslyassemble into liposome particles. When the drug API is carried by thelipid solution or by aqueous solution, it may be encapsulated in theliposome by either lipophilic or electrostatic interaction, or both,between the API and the lipids.

By Diffusion

The present invention also provides a method of producing LNP that donot contain an API (so-called “empty” LNP). In such embodiments, the APIis absent from both the lipid solution and the aqueous solution that aremixed in the manifold. The API may be loaded into the liposomes by theprocess of diffusion or another process. For example, doxorubicin may beloaded into the liposome with a pH gradient. See U.S. patent applicationSer. No. 10/019,200, PCT Publication No. WO 2001/005373, U.S. Pat. Nos.5,785,987, 5,380,531, 5,316,771, and 5,192,549, all of which areincorporated herein by reference.

Preferably, the API is mixed with a LNP solution to upload the API intothe liposome by diffusion. In one aspect, the API is dissolved in anaqueous solution, and the solution is mixed with the empty LNP. Inanother aspect, the API may be readily soluble in the solution of emptyLNP, and therefore, the API may be directly mixed with the solution ofthe empty LNP.

The volume ratio of the solution of the API to the empty liposomesolution of the API is preferably in the range from about 1:50 to about1:5. A lower volume of the solution is preferred because it avoids asignificant dilution to the final liposome solution.

The drug encapsulation efficiency is preferably greater than 70%. Morepreferably the efficiency is greater than 80%. Most preferably, theefficiency is greater than 90%.

Liposome Concentration Adjustment

Tangent flow filtration may be used to concentrate the liposomesolution.

Buffer Change

Residual organic solvent in the LNP solution may be removed by a bufferchange. Preferably, the buffer change is performed by tangent flowfiltration. In another embodiment, the buffer change may be performed bydialysis.

Sterile Filtration

The liposome solutions are preferably sterilized by passing a 0.22micron sterile filter.

US patents, patent applications, PCT publications that describe the useof LNP are: U.S. Pat. No. 8,067,390, PCT Publication No. WO 02/100435A1,PCT Publication No. WO 03/015757A1, PCT Publication No. WO 04/029213A2;U.S. Pat. No. 5,962,016, U.S. Pat. No. 5,891,467, U.S. Pat. No.5,030,453, and U.S. Pat. No. 6,680,068; and US Patent ApplicationPublication No. 2004/0208921, all of which are incorporated herein byreference.

The following examples are illustrative and not restrictive. Manyvariations of the technology will become apparent to those of skill inthe art upon review of this disclosure. The scope of the technologyshould, therefore, be determined not with reference to the examples, butinstead should be determined with reference to the appended claims alongwith their full scope of equivalents.

EXAMPLES Materials

All the manifolds used in the examples were made of PEEK polymer andwere purchased from a commercially available source.

Methods Example 1 Preparation of Liposomes with Doxil Lipid Composition

The lipids were dissolved in anhydrous ethanol. Aqueous Solutions 1, 2,3 were all 250 mM ammonium sulfate, pH 6.5. The composition of the lipidsolution is illustrated in the Table of Example 1. The molar ratio ofthe lipids is substantially the same as the formulation of Doxil whichis a clinically used anti-cancer liposome formulation of doxorubicin.One milliliter each of above 4 solutions was loaded into a 20 mlsyringe; each syringe was connected to an inlet port of a 5-portmanifold by a tubing. Through the tubing, the solutions in the syringeswere pumped into the mixing chamber of the manifold by a syringe pump.The pore size (diameter) of the manifold was 1.0 mm or 1.6 mm. The flowrate of the mixing was 5, 10, or 20, or 30, or 40, or 50 ml/min. Theliposome solution exited through the outlet port and was collected in aglass vial.

The particle size and polydispersity index were determined by MalvernZetasizer Nano ZS in HEPES buffered saline (10 mM HEPES, pH 7.4, 138 mMNaCl). The results are presented in FIG. 4A, FIG. 4B, FIG. 4C and FIG.4D.

Lipid Composition of Example 1

Lipid % (molar) mg/ml Hydrogenated Soy PC 56.5 17.24 Cholesterol 38.05.76 mPEG2000-DSPE 5.3 5.76

Example 2 Preparation of Doxorubicin Loaded Liposomes

The lipids were dissolved in anhydrous ethanol. Aqueous Solutions 1, 2,3 were all 250 mM ammonium sulfate, pH 6.5. The composition of the lipidsolution are illustrated in the Table of Example 2. The molar ratio ofthe lipids is substantially the same as the formulation of Doxil whichis an anti-cancer liposomal formulation of doxorubicin. One millilitereach of above 4 solutions was loaded into a 20 ml syringe; each syringewas connected to an inlet port of a five-port manifold by a tubing.Through the tubing, the solutions in the syringes were pumped into themixing chamber of the manifold by a syringe pump. The pore size(diameter) of the mixer was 0.5 mm, and the flow rate was 40 ml/min. Theliposome solution exited through the outlet port and was collected in aglass vial. The buffer was changed into histidine/sucrose buffer (12.5mM histidine, 9.2% sucrose, pH 6.5) by dialysis. The Z-average particlesize was 86.1 nm with PDI of 0.021.

Two milliliters of empty liposomes was mixed with 0.198 ml ofdoxorubicin solution at a concentration of 10 mg/ml in histidine/sucrosebuffer. and incubated at 42° C. for 2 hours. The lipid/doxorubicin ratio(w/w) was 7.99, 99.5% of doxorubicin was loaded into the liposome. TheZ-average particle size of the loaded liposome was 87.3 nm with PDI of0.032. The Cryo-TEM images of doxorubicin-loaded liposomes made by thismethod was shown in FIG. 5.

Lipid Composition of Example 2

% (molar) mg/ml Hydrogenated Soy PC 56.5 17.24 Cholesterol 38.0 5.76mPEG2000-DSPE 5.3 5.76

Example 3 Preparation of Liposomes Using a 6-Port Manifold

The lipids were dissolved in anhydrous ethanol. One milliliter of lipidsolution was loaded into a 5-ml syringe, the ammonium sulfate solution(250 mM, pH 6.5) was loaded into four 5-ml syringes with 1 ml for eachsyringe. Each syringe was connected to an inlet port of a 6-portmanifold (IDEX Health & Sciences, part# P-152) by a tubing. The lipidsand the ammonium sulfate solutions loaded in the syringes were pumpedinto the mixing chamber of the manifold by a syringe pump. The pore sizeof the 6-port manifold was 1.0 mm and the flow rate was 20 ml/min. Theliposome solution exited through the outlet port and was collected in aglass vial.

The Z-average particle size and polydispersity index determined byMalvern Zetasizer Nano ZS in HEPES buffered saline (10 mM HEPES, pH 7.4,138 mM NaCl) were 80.2 nm and 0.207, respectively.

Lipid Composition of Example 3

% (molar) mg/ml Hydrogenated Soy PC 56.5 17.24 Cholesterol 38.0 5.76mPEG2000-DSPE 5.3 5.76

Example 4 Preparation of Liposomes Using a 7-Port Manifold

The lipids were dissolved in anhydrous ethanol. One milliliter of lipidsolution was loaded into a 5-ml syringe, the ammonium sulfate solution(250 mM, pH 6.5) was loaded into five 5-ml syringes with 1 ml for eachsyringe. Each syringe was connected to an inlet port of a 7-portmanifold (IDEX Health & Sciences, part# P-150) by a tubing. The lipidsand the ammonium sulfate solutions loaded in the syringes were pumpedinto the mixing chamber of the manifold by a syringe pump. The pore sizeof the 7-port manifold was 1.0 mm and the flow rate was 20 ml/min. Theliposome solution exited through the outlet port and was collected in aglass vial

The Z-average particle size and polydispersity index determined byMalvern Zetasizer Nano ZS in HEPES buffered saline (10 mM HEPES, pH 7.4,138 mM NaCl) were 60.1 nm and 0.120, respectively.

Lipid Composition of Example 4

% (molar) mg/ml Hydrogenated Soy PC 56.5 17.24 Cholesterol 38.0 5.76mPEG2000-DSPE 5.3 5.76

Example 5 Preparation of Liposomes Using a 9-Port Manifold

The lipids were dissolved in anhydrous ethanol. One milliliter of lipidsolution was loaded into a 5-ml syringe, the ammonium sulfate solution(250 mM, pH 6.5) was loaded into seven 5-ml syringes with 1 ml for eachsyringe. Each syringe was connected to an inlet port of a 9-portmanifold (IDEX Health & Sciences, part# P-191) by a tubing. The lipidsand the ammonium sulfate solutions loaded in the syringes were pumpedinto the mixing chamber of the manifold by a syringe pump. The pore sizeof the 9-port manifold was 1.0 mm, and the flow rate was 20 ml/min. Theliposome solution exited through the outlet port and was collected in aglass vial

The Z-average particle size and polydispersity index determined byMalvern Zetasizer Nano ZS in HEPES buffered saline (10 mM HEPES, pH 7.4,138 mM NaCl) were 63.1 nm and 0.133, respectively.

Lipid Composition of Example 5

% (molar) mg/ml Hydrogenated Soy PC 56.5 17.24 Cholesterol 38.0 5.76mPEG2000-DSPE 5.3 5.76

Example 6 Preparation of siRNA Liposomes

Lipid solution: The components of the lipids solution was illustrated inthe Table of Example 6.

The RNA was siApoB-1 sequence as described in 61/791,054 in Example 6.

Aqueous Solution 1: siRNA: 0.5 mg/ml in a citrate buffer (20 mM, pH5.0); Aqueous Solution 2: 20 mM citrate, pH 5.0, 100 mM NaCl; AqueousSolution 3: same as Solution 2

One milliliter of each of above 4 solutions was loaded into a 20 mlsyringe; each syringe was connected to an inlet port of a 5-portmanifold by a tubing. The lipids, siRNA, and the aqueous buffersolutions loaded in the syringes were pumped into the mixing chamber ofthe manifold by a syringe pump. The liposome solution exited through theoutlet port and was collected in a glass vial The pore size of the5-port manifold mixer was 1 mm or 1.6 mm. The flow rate was 5, or 10, or30, or 40, or 50 ml/min.

The particle size and PDI of siRNA liposomes were determined by MalvernZetasizer Nano ZS in HEPES buffered saline (10 mM HEPES, pH 7.4, 138 mMNaCl), The particle morphology was imaged by Cryo-TEM. The results areshown in FIG. 6. As shown in the figure, lipids form unilamellarliposomes, in which doxorubicin forms crystals.

Lipid Solution in Anhydrous Ethanol of Example 6

% (molar) mg/ml 1,2-dilinoleyloxy-3- 38.7 2.775 dimethylaminopropane(DLinDMA) Cholesterol 46.4 2.095 DSPC 13.0 1.200 mPEG2000-DMA 1.9 0.570

Example 7 Preparation of Liposome and Lipid Discs from the SameFormulation by Altering the Flow Rate

The lipids were dissolved in anhydrous ethanol. Aqueous Solutions 1, 2,3 were all 250 mM ammonium sulfate, pH 6.5. The composition of the lipidsolution are illustrated in the Table of Example 7. One milliliter eachof the above 4 solutions was loaded into a 20 ml syringe; each syringewas connected to an inlet port of a five-port manifold by tubing.Through the tubing, the solutions in the syringes were pumped into themixing chamber of the manifold by a syringe pump. The pore size(diameter) of the manifold was 1.0 mm, and the flow rate was 40 ml/min,or 5 ml/min. The liposome or lipid disc solution exited through theoutlet port and was collected in a glass vial. The buffer was changedinto HEPES buffer (10 mM HEPES, 138 mM NaCl, pH 7.5) by dialysis. Theliposome was loaded with doxorubicin. The Cryo-TEM imaging identifiedthat the 40 ml/min flow rate generated unilamellar liposomes (FIG. 7A)having a Z-average particle size of 86.1 nm and a PDI of 0.021. The 5.0ml/min flow rate predominantly generated lipids discs (FIG. 7B) withabout 60 nm in diameter and about 6 nm lipid bilayer thickness.

Lipid Composition of Example 7

% (Molar) mg/ml Hydrogenated Soy PC 56.6 21.9 Cholesterol 38.4 7.3mPEG2000-DSPE 5.0 7.3

Example 8 The Effects of Number and Position of the Exit Ports onLiposome Particle Size

The lipids were dissolved in anhydrous ethanol. Aqueous Solutions 1, 2,3 were all 250 mM ammonium sulfate, pH 6.5. The composition of the lipidsolution is illustrated in the Table of Example 7. One milliliter eachof above 4 solutions was loaded into a 20 ml syringe; each syringe wasconnected to an inlet port of a seven-port manifold by tubing(configured variously as shown in FIGS. 8A-8D). Through the tubing, thesolutions in the syringes were pumped into the mixing chamber of themanifold by a syringe pump. The pore size (diameter) of the manifold was0.5 mm, and the flow rate was 35 ml/min. One (1) or two (2) or three (3)of the rest 3 ports (the center one perpendicular to other ports and twoside ports, see the illustration in FIG. 8) was (were) used as theoutlet port(s) for the liposome solution. The liposome solution exitedthrough the outlet port(s) and was collected in a glass vial. Thedifferent number of outlet ports resulted in different liposome particlesizes: 91 nm (PDI 0.146) for 3 ports of outlet (FIG. 8A), 81 nm (PDI0.089) for two ports outlet (FIG. 8B); and 74-75 nm (PDI 0.052-0.088)for one port outlet (FIGS. 8C and 8D). The position of the outlet had nosignificant effects on the particle size (FIGS. 8C and 8D). Therefore,the liposome particle size can be controlled via the numbers of theoutlet ports.

Lipids Composition of Example 8

% (Molar) mg/ml Hydrogenated Soy PC 56.6 30.8 Cholesterol 38.4 10.3mPEG2000-DSPE 5.0 10.3

1. A method for preparing lipid nanoparticles (LNP), the methodcomprising: a) introducing i) one or more streams of a lipid solutionvia a first set of one or more inlet ports of a manifold and ii) one ormore streams of an aqueous solution via a second set of one or more aninlet ports of the manifold, thereby mixing the lipid solution and theaqueous solution so as to produce an LNP solution; and b) recovering theLNP solution via one or more outlet ports of the manifold; wherein theangle between at least one lipid and at one aqueous solution inlet portsis not 180° or a substantially similar angle.
 2. The method of claim 1,wherein the angle between at least one lipid and at one aqueous solutioninlet ports is about 120° or less.
 3. The method of claim 1, wherein theaqueous solution in step ii) is introduced via at least two inlet ports.4. The method of claim 3, wherein the aqueous solution in step ii) isintroduced via at least 3 but no more than 11 inlet ports.
 5. The methodof claim 3, wherein at least two aqueous inlet ports and at least onelipid solution inlet port are in the same plane.
 6. The method of claim5, wherein at least one outlet port is substantially perpendicular tothe plane of inlet ports.
 7. The method of claim 5, wherein at least oneoutlet port is substantially not perpendicular to the plane of inletports.
 8. The method of claim 3, wherein at least two aqueous solutioninlet ports and at least on lipid solution inlet port are not in thesame plane.
 9. The method of claim 1, wherein the lipid solution and/orthe aqueous solution is introduced at a flow rate of about 1 ml/min to6,000 ml/min.
 10. The method of claim 9, wherein the lipid solutionand/or the aqueous solution is introduced via port size of 0.1-5 mm at aflow rate about 1 ml/min to about 2,500 ml/min.
 11. The method of claim1, wherein the flow velocity of the lipid solution and/or the aqueoussolution is the lipid solution and/or the aqueous solution is from about0.002 m/s to about 10 m/s.
 12. The method of claim 1, wherein the LNPsolution comprises substantially lipid discs.
 13. The method of claim 1,wherein the LNP solution comprises substantially liposomes.
 14. Themethod of claim 1, wherein the mean particle size of LNP is from about10 nm to about 2,000 nm.
 15. The method of claim 1, wherein the LNP havea polydispersity index from about 0.0005 to about 0.5.
 16. The method ofclaim 1, wherein the lipid solution comprises lipids dissolved in anorganic solvent.
 17. The method of claim 1, wherein the organic solventis ethanol at a concentration of about 70% or more.
 18. The method ofclaim 16, wherein the concentration of total lipids in the lipidsolution is in the range from about 1 mg/ml to about 200 mg/ml.
 19. Themethod of claim 1, wherein one of the lipids in the lipid solution ischosen from anionic lipid, cationic lipid, or neutral lipid.
 20. Themethod of claim 3, wherein the aqueous solution introduced into at leastone of the inlet ports differs from a second aqueous solution introducedinto another inlet port.
 21. The method of claim 1, wherein the aqueoussolution and/or the lipid solution comprises an active pharmaceuticalingredient (API).
 22. The method of claim 1, wherein step a) furthercomprises introducing iii) one or more streams of non-aqueous solutionsvia one or more inlet ports of the manifold.
 23. The method of claim 1,further comprising step c) loading LNP recovered from the LNP solutionwith an API.
 24. The method of claim 21 or claim 23, wherein the API isa small molecule, a peptide, a protein, RNA, or DNA.
 25. The method ofclaim 21 or claim 23, wherein the API is an anticancer agent, anantibiotic agent, an antiviral agent, an anti-fungal agent, or ananalgesic.
 26. The method of claim 25, wherein the anti-cancer agent ischosen from daunorubicin, doxorubicin, paclitaxel, docetaxel, cisplatin,carboplatin, cytarabine, floxuridine, fludarabine, fluorouracil,iproplatin, leuprolide acetate, and methotrexate.
 27. The LNP solutionmade by the method of claim
 1. 28. A pharmaceutical composition preparedusing the LNP solution of claim
 26. 29. A device adapted to perform themethod of claim 1.